Mechanisms of Norovirus Pathogenesis and Replication to Develop Therapeutics Project Summary Noroviruses are the most common cause of gastroenteritis in the United States, and they cause a significant economic and health burden to the population. A significant barrier to progress in making effective therapeutics, antivirals and vaccines to control human norovirus illness was the previous lack of a cell culture system. The focus of this program project is to build upon previous basic science and translational findings and discoveries to allow continued progress in our understanding of the biology and pathogenesis of these viruses so that improved control strategies can be developed. We recently developed a successful replication system that discovered strain- specific requirements for replication and allows neutralization assays and antivirals to be tested. We will pursue key questions on virus pathogenesis and cellular responses, structure-function studies on the interactions between virus proteins and host components, discover key factors that restrict extensive propagation and test antivirals and neutralizing antibodies in three separate projects. In Project 1 we will perform studies designed to provide us with a fundamental molecular understanding of how human noroviruses cause disease, and of what epitopes are recognized by human sera that are associated with virus neutralization, virus clearance, and protective immunity. In addition, we will build on recent success and continue to develop antivirals for treatment and prevention strategies that can be applied to at risk populations. In Project 2 we will continue to improve the cultivation system, focusing on identifying the cell receptor(s) and understanding why all HuNoV strains do not grow in the replication system. We will also evaluate molecular mechanisms by which norovirus replication regulates cellular innate responses and whether these cellular responses regulate viral replication and spread. In Project 3 we will determine the structural basis for novel functions of key proteins that regulate viral replication and virus-host interactions to provide a rational framework for the development of antivirals. These projects will be supported by three cores. Core A (Administrative Core) will provide centralized administrative and fiscal management support and will coordinate programmatic activities. Core B (the Microscopy and Enteroid Core) will provide expertise and services to each project related to electron microscopy and fluorescent microscopy. This core will also maintain and provide enteroids, including genetically-modified cultures and biosensor lines to all projects. Core C (the Protein and Small Molecule Chemistry Core) will provide all projects with access to purified proteins and virus-like particles as well as facilitating site-directed mutagenesis activities needed. In addition, the Core will synthesize small molecules to be used for protease and polymerase inhibition studies in each project. The program project brings together a highly collaborative group of investigators with diverse skills and talents, and an substantial history of working together. As in the previous funding period, the interactions among each project and each of the cores will be extensive and synergistic such that the activities of each project will be enhanced considerably over what could be accomplished if the projects were pursued independently.